Chang, J. J.-Y. | bioRxiv Science

bioRxiv2023

Uncovering strain- and age- dependent differences in innate immune response to SARS-CoV-2 infection in nasal epithelia using combined short and long-read scRNA-seq

Assessing the impact of SARS-CoV-2 variants on the host is crucial with continuous emergence of new variants. We employed single-cell sequencing to investigate host transcriptomic response to ancestral and Alpha-strain SARS-CoV-2 infections within air-liquid-interface human nasal epithelial cells from adults and adolescents. Strong innate immune responses were observed across lowly-infected and bystander cell-types, and heightened in Alpha-infection. Contrastingly, the innate immune response of highly-infected cells was like mock-control cells. Alpha highly-infected cells showed increased expression of protein refolding genes compared with ancestral-strain-infected adolescent cells. Oxidative phosphorylation- and translation-related genes were down-regulated in bystander cells versus infected and mock-control cells, suggesting that the down-regulation is protective and up-regulation supports viral activity. Infected adult cells revealed up-regulation of these pathways compared with infected adolescents, implying enhanced pro-viral states in infected adults. Overall, this highlights the complexity of cell-type-, age- and viral-strain-dependent host epithelial responses to SARS-CoV-2 and the value of air-liquid-interface cultures.

immunology↗

bioRxiv2021

Long-read RNA sequencing identifies polyadenylation elongation and differential transcript usage of host transcripts during SARS-CoV-2 in vitro infection

Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) combined with the Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analysed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~136 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.

immunology↗